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Figure 1. Schematic drawings of the experimental procedures. A: Fate maps adapted from Tschumi (1957). The zone of polarizing activities (Cameron and Fallon, 1977) are combined with the maps. The high and low polarizing activities represent the percentages of digit duplications after a 180-degree rotation (anteroposterior [AP] and dorsoventral [DV] axes were opposed) more than 40% and 20%, respectively. The photograph shows the skeletal preparation of a normal limb at stage 60. The arrowheads indicate anterior digits with a claw. All limbs are left hindlimbs and dorsal view. pe, pelvis; fe, femur; ft, fibula and tibia; ta, tarsus; met, metatarsus; “12345,†digits; A, anterior; P, posterior; Pro, proximal; Dis, distal; st, stage. B: Amputation levels for normal regeneration. The limb buds were amputated at the distal fourth of the presumptive femur level (st 51–54) or the middle of the presumptive tarsus level (stage 52–54). The amputation levels of stage 54 were not presented in this figure. C: Rotation of limb buds. The limb buds were amputated at the presumptive femur or tarsus level and grafted to the stumps. The left (L) and right (R) limbs are represented by white and gray, respectively. Stage 52 limb buds are shown in this figure. D, dorsal; V, ventral. D: Induction of intercalary limb regeneration. The limb bud tips were prepared by the amputation of limb buds at the presumptive tarsus level and transplanted to the stumps that were amputated at the presumptive femur level. The presumptive zeugopodium (fibula and tibia) were discarded. Stage 52 limb buds are shown in this figure. E: Transplantation of blastemas. Formation of blastemas was induced by the amputation of the stage 52 limb bud at the presumptive tarsus level. Five days after amputation (the tadpole developed to stage 53), the blastema was transplanted to the stage 52 stump, which was amputated at the presumptive femur or tarsus level. The dot indicates the posterior side of blastema.
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Figure 2. Digit pattern of regenerates from limb buds amputated at various stages. The limb buds were amputated at the presumptive femur or tarsus level. The digit pattern was examined after metamorphosis by observing the skeletal preparations. The total number of each operation is shown in parentheses. P, posterior type digit; A, anterior type digit.
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Figure 3. Supernumerary regeneration induced by the rotation of the limb bud tip. The limb bud was amputated and rotated at stage (st) 52 (A–D) or stage 53 (E,F). The transplantation procedure is shown in Figure 1C. A and E, B and F, C, and D correspond to C1, C4, C2, and C5 in Fig. 1C, respectively. “Double supernumerary limbs†indicates the limb with two sets of supernumerary digits generated at both the anterior and posterior sides, as seen in A. “Single supernumerary limb†indicates the limb with a single set of supernumerary digits generated at either the anterior or posterior, as seen in B–F. The red arrowheads indicate the amputation plane. The black arrowheads and arrows represent each set of supernumerary digits. Asterisks indicate supernumeraries. fe, femur; ft, fibula and tibia; ta, tarsus; “12345,†digits; AP, anteroposterior; DV, dorsoventral. Scale bar = 2 mm in A (applies to A–F).
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Figure 4. Intercalary regeneration. The limb bud tip (amputated at the presumptive tarsus level) was transplanted to the stump (amputated at the presumptive femur level). The presumptive zeugopodium (fibula and tibia) were discarded. Stage (st) 52 (A,D,F), stage 53 (B,E,G), and stage 54 (C) tadpoles were used for this experiment. The transplantation procedure is shown in Figure 1D. A–C and D,E correspond to D1 and D3 in Fig. 1D, respectively. The control right limbs (F,G) and operated left limbs (D,E) are the same animals, respectively. The red arrowheads indicate the amputation plane. The black arrows represent supernumerary digits. Asterisks indicate the regenerated fibula and tibia. fe, femur; ft, fibula and tibia; ta, tarsus; “12345,†digits; AP, anteroposterior; DV, dorsoventral. Scale bar = 2 mm in A (applies to A–G).
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Figure 5. A–D: Transplantation of blastema to the stage 52 stump. Blastema was induced by the amputation of the stage 52 limb bud at the presumptive tarsus level. Five days after amputation, blastema was grafted to the stage 52 stump amputated at the presumptive femur (A,B) or tarsus (D) level with (B,D) or without (A) 180-degree rotation. The control right limb (C) and operated left limb (B) are the same animal. The transplantation procedure is shown in Figure 1E. A, B, and D correspond to E1, E2, and E3 in Figure 1E, respectively. The red arrowheads indicate the amputation plane. The black arrowheads and arrows represent each set of supernumerary digits. Asterisks indicate the regenerated fibula and tibia. fe, femur; ft, fibula and tibia; ta, tarsus; “12345,†digits; AP, anteroposterior; DV, dorsoventral. Scale bar = 2 mm in A (applies to A–D).
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Figure 6. Fibroblast growth factor-8 (FGF-8) -induced intercalary regeneration. The limb buds at stage (st) 52 (A–C) or stage 53 (D,E) were amputated at the presumptive tarsus level and transplanted to the stump that was amputated at the presumptive femur level (same operation as in Fig. 1D, D1). The bead soaked in FGF-8 was held with tungsten wire pins between the limb bud tip and the stump. A: The pins were removed two days after the operation. B,D: The left limb regenerated with FGF-8 bead. C,E: Control right limb (without operation). The manipulated and control limbs belong to the same frog. The red arrowheads indicate amputation plane. The black arrows in A,D point to a bead. D: The implanted bead was sometimes observed in the skeletal preparation and always found at the amputation plane of the femur level. Asterisks indicate the intercalated skeletons. fe, femur; ft, fibula and tibia; ta, tarsus; “12345,†digits. Scale bars = 400 μm in A, 2 mm in B (applies to B–E).
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Figure 7. Expression of fgf-8 expanded in the limb bud with a 180-degree rotation. The stage 52 limb bud tip (amputated at the presumptive tarsus level) was transplanted to the stump amputated at the presumptive femur level with (B) or without (D) 180-degree rotation. Five days after the operation, the samples were processed for hybridization with fgf-8 cDNA. Manipulated (B,D) and control (A,C) limbs are left and right, respectively. The black arrows indicate the normal and expanded expression of fgf-8 in apical ectodermal ridge and the black arrowheads in (B) indicate the ectopic expression of fgf-8 in the stump. The red arrowheads indicate the amputation plane. a, anterior; p, posterior. Scale bar = 200 μm in D (applies to A–D).
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